A Monoclonal Antibody Against Human Vitamin-D-binding Protein for the Analysis of Genetic Variation in the Group-specific Component System (Gc)

Overview

Journal Hum Genet

Specialty Genetics

Date 1990 Jan 1

PMID 2298449

Authors

R Hoffmann

A Braun

H CLEVE

Affiliations

Soon will be listed here.

Abstract

We have developed a murine hybridoma cell line that is stable in secreting a monoclonal antibody (hDBP-1) directed against the group-specific component (Gc) molecule. The hDBP-1 is monospecific for Gc and does not crossreact with human albumin, which has 23% of its amino acid residues identical with vitamin-D-binding protein (DBP). The subclass of the antibody is IgG1 for the heavy chain, the light chain being of the kappa type. Isoelectric focusing discloses four major bands for the hDBP-1 with isoelectric points between pH 6.5 and 7.8. Binding to the antigen at different pH values was determined: there is high affinity in the physiological range and no binding at pH 3.5 and lower. In the presence of high salt concentrations, binding was reduced to about 50% at 1.5 M NaCl. The hDBP-1 recognizes the common human Gc types and the Gc of all apes and old world monkeys. No reaction was observed with the Gc of other mammals such as horses, cattle, rats, rabbits, sheep, goats and pigs. By testing hDBP-1 against 77 of the more than 120 known rare human Gc variants, it could be shown that this monoclonal antibody cannot recognize seven of these rare variants and can only poorly recognize nine. The binding site of hDBP-1 to Gc is not related to the binding site of Gc with G-actin: it recognizes Gc, the binary complex between Gc and G-actin, as well as the ternary complex between Gc, G-actin and DNase I. Competition assays with vitamin D3 and Gc in enzyme-linked immunosorbent assay indicate that the epitope of hDBP-1 on the Gc molecule may be related to the vitamin-D3-binding site.

References

Cooke N . Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA. J Biol Chem. 1986; 261(7):3441-50. View

Sinclair D, Parrott D, Stott D . Quantitation of monoclonal immunoglobulins by immuno-isoelectric focusing and its application for monitoring secretory B cell neoplasia. J Immunol Methods. 1986; 90(2):247-55. DOI: 10.1016/0022-1759(86)90082-7. View

Schoentgen F, Metz-Boutigue M, Jolles J, Constans J, Jolles P . Complete amino acid sequence of human vitamin D-binding protein (group-specific component): evidence of a three-fold internal homology as in serum albumin and alpha-fetoprotein. Biochim Biophys Acta. 1986; 871(2):189-98. DOI: 10.1016/0167-4838(86)90173-1. View

Hamilton R, Reimer C, Rodkey L . Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis. Hybridoma. 1987; 6(2):205-17. DOI: 10.1089/hyb.1987.6.205. View

Pierce E, Dame M, Bouillon R, Van Baelen H, DeLuca H . Monoclonal antibodies to human vitamin D-binding protein. Proc Natl Acad Sci U S A. 1985; 82(24):8429-33. PMC: 390929. DOI: 10.1073/pnas.82.24.8429. View

De Blas A, Ratnaparkhi M, MOSIMANN J . Estimation of the number of monoclonal hybridomas in a cell-fusion experiment. Methods Enzymol. 1983; 92:36-9. DOI: 10.1016/0076-6879(83)92007-4. View

Constans J, Viau M . Group-specific component: evidence for two subtypes of the Gc1 gene. Science. 1977; 198(4321):1070-1. DOI: 10.1126/science.73222. View

Haddad J, Kowalski M, Sanger J . Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins). Biochem J. 1984; 218(3):805-10. PMC: 1153408. DOI: 10.1042/bj2180805. View

CLEVE H, Constans J . The mutants of the vitamin-D-binding protein: more than 120 variants of the GC/DBP system. Vox Sang. 1988; 54(4):215-25. DOI: 10.1111/j.1423-0410.1988.tb03908.x. View

10.

THOMAS Jr W, MORGAN H, Connor T, Haddock L, BILLS C, Howard J . Studies of antiricketic activity in sera from patients with disorders of calcium metabolism and preliminary observations on the mode of transport of vitamin D in human serum. J Clin Invest. 1959; 38(7):1078-85. PMC: 293255. DOI: 10.1172/JCI103884. View

11.

Cooke N, David E . Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family. J Clin Invest. 1985; 76(6):2420-4. PMC: 424397. DOI: 10.1172/JCI112256. View

12.

Van Baelen H, Bouillon R, DE MOOR P . Vitamin D-binding protein (Gc-globulin) binds actin. J Biol Chem. 1980; 255(6):2270-2. View

13.

Bouillon R, Van Baelen H, ROMBAUTS W, DE MOOR P . The purification and characterisation of the human-serum binding protein for the 25-hydroxycholecalciferol (transcalciferin). Identity with group-specific component. Eur J Biochem. 1976; 66(2):285-91. DOI: 10.1111/j.1432-1033.1976.tb10518.x. View

14.

Haddad Jr J, Walgate J . 25-Hydroxyvitamin D transport in human plasma. Isolation and partial characterization of calcifidiol-binding protein. J Biol Chem. 1976; 251(16):4803-9. View

15.

Perez H, Kelly E, Chenoweth D, Elfman F . Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin). J Clin Invest. 1988; 82(1):360-3. PMC: 303517. DOI: 10.1172/JCI113595. View

16.

Coue M, Constans J, Viau M, OLOMUCKI A . The effect of serum vitamin D-binding protein on polymerization and depolymerization of actin is similar to the effect of profilin on actin. Biochim Biophys Acta. 1983; 759(3):137-45. DOI: 10.1016/0304-4165(83)90305-7. View

17.

Schoentgen F, Metz-Boutigue M, Jolles J, Constans J, Jolles P . Homology between the human vitamin D-binding protein (group specific component), alpha-fetoprotein and serum albumin. FEBS Lett. 1985; 185(1):47-50. DOI: 10.1016/0014-5793(85)80738-9. View

18.

Pollard T, Cooper J . Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986; 55:987-1035. DOI: 10.1146/annurev.bi.55.070186.005011. View

19.

Perez H, Chenoweth D, Goldstein I . Attachment of human C5a des Arg to its cochemotaxin is required for maximum expression of chemotactic activity. J Clin Invest. 1986; 78(6):1589-95. PMC: 423925. DOI: 10.1172/JCI112751. View

20.

Hirschfeld J . Immune-electrophoretic demonstration of qualitative differences in human sera and their relation to the haptoglobins. Acta Pathol Microbiol Scand. 1959; 47:160-8. DOI: 10.1111/j.1699-0463.1959.tb04844.x. View